International Journal of Molecular Sciences Review STIM Proteins: An Ever-Expanding Family Herwig Grabmayr , Christoph Romanin * and Marc Fahrner * Institute of Biophysics, Johannes Kepler University Linz, Gruberstrasse 40, 4020 Linz, Austria; [email protected] * Correspondence: [email protected] (C.R.); [email protected] (M.F.) Abstract: Stromal interaction molecules (STIM) are a distinct class of ubiquitously expressed single- pass transmembrane proteins in the endoplasmic reticulum (ER) membrane. Together with Orai ion channels in the plasma membrane (PM), they form the molecular basis of the calcium release- activated calcium (CRAC) channel. An intracellular signaling pathway known as store-operated calcium entry (SOCE) is critically dependent on the CRAC channel. The SOCE pathway is activated by the ligand-induced depletion of the ER calcium store. STIM proteins, acting as calcium sensors, subsequently sense this depletion and activate Orai ion channels via direct physical interaction to allow the influx of calcium ions for store refilling and downstream signaling processes. This review article is dedicated to the latest advances in the field of STIM proteins. New results of ongoing investigations based on the recently published functional data as well as structural data from nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations are reported and complemented with a discussion of the latest developments in the research of STIM protein isoforms and their differential functions in regulating SOCE. Keywords: STIM1; STIM2; isoforms; Orai; CRAC; SOCE; CC1; NMR; structure; simulation 1. Introduction Chemical elements in their ionic form are indispensable factors for the correct function Citation: Grabmayr, H.; Romanin, C.; of vital cells and organisms. Among the various ions that have been selected in the course Fahrner, M. STIM Proteins: An of evolution to be involved in living organisms, calcium (Ca2+) occupies a special place. Ever-Expanding Family. Int. J. Mol. Besides the enormous amounts found in bones and teeth, calcium plays an outstanding Sci. 2021, 22, 378. https://doi.org/ role as a second messenger in every cell [1–4]. In a resting cell, calcium is present in a very 10.3390/ijms22010378 low cytosolic concentration. For intracellular calcium-dependent signal transduction, an Received: 5 December 2020 increase of the cytosolic calcium concentration is necessary. This is achieved by calcium Accepted: 26 December 2020 influx from the extracellular space or by depletion of the intracellular calcium store in the Published: 31 December 2020 endoplasmic reticulum (ER) [4]. Among the different calcium-selective transmembrane proteins, the pathway of store-operated calcium entry (SOCE) plays an important role. Publisher’s Note: MDPI stays neu- Two key proteins, stromal interaction molecule (STIM) and Orai, form the calcium release- tral with regard to jurisdictional clai- activated calcium (CRAC) channel system that mediates SOCE and is responsible for ms in published maps and institutio- regulated calcium influx in many cell types [5–8]. Orai is the calcium-selective channel in nal affiliations. the plasma membrane (PM) and STIM is the calcium sensor in the ER membrane. Ligand binding to the extracellular surface of the cell leads to cytosolic activation of phospholipase C (PLC), which in turn cleaves the head group of a specific phospholipid to form cytosolic inositol trisphosphate (IP3) and membrane-bound diacylglycerol (DAG). IP3 diffuses Copyright: © 2020 by the authors. Li- throughout the cytosol to IP3 receptors in the ER membrane and elicits depletion of the censee MDPI, Basel, Switzerland. store. As a result, the ER luminal calcium concentration decreases dramatically [2,4,9] This article is an open access article This is the step that represents the STIM-activating signal [10–13]. STIM has, among other distributed under the terms and con- ditions of the Creative Commons At- elements, an EF hand in its ER luminal N-terminus with which the calcium concentration in tribution (CC BY) license (https:// the ER can be sensed [14,15]. Lowering the ER calcium concentration results in dissociation creativecommons.org/licenses/by/ of calcium from the STIM EF hand and thus in a conformational change of the STIM 4.0/). N-terminus [10]. The signal propagates across the transmembrane (TM) domain to the Int. J. Mol. Sci. 2021, 22, 378. https://doi.org/10.3390/ijms22010378 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 378 2 of 18 cytosolic C-terminus of STIM [16]. A cascade of conformational changes occurs at the STIM C-terminus, resulting in oligomerization and spatial extension of the protein [16–22]. The extended STIM protein translocates to the cell periphery and interacts directly with the PM-resident protein Orai [8,23,24]. The physical coupling to Orai occurs mainly at its C-terminus; however, interactions with Orai loop2 and N-terminus are also involved in the correct gating of the channel [25,26]. In its monomeric form, Orai has 4 TM domains, a cytosolic N- and C-terminus, and a cytosolic loop2 between TM2 and TM3. Six Orai monomers form the functional hexameric Orai channel [27]. Six TM1 domains constitute the calcium-selective channel pore, which is separated from the hydrophobic milieu of the PM and from the TM4 domains by a ring consisting of TM2 and TM3. The Orai N-terminus merges into the TM1 and the C-terminus merges into the TM4 [8,25,27]. The importance of SOCE for physiological cytosolic calcium homeostasis and the calcium-dependent function of critical cell-biological processes is underlined by several gain- (GoF) and loss-of-function (LoF) mutations within STIM and Orai. GoF mutations raise the intracellular calcium concentration by eliciting constitutive CRAC channel activation as well as SOCE. This leads to a clinical continuum including disease phenotypes termed York platelet syndrome, Stormorken syndrome, and tubular aggregate myopathy. Pathological manifestations thereby include (but are not limited to) myopathy, thrombocytopenia, miosis, ichthyosis, and dyslexia. In contrast, LoF mutations abolish CRAC channel activation. The absence of SOCE results in severe combined immunodeficiency, autoimmunity, ectodermal dysplasia, and muscular hypotonia [28,29]. In this review, we focus on the growing family of STIM isoforms, which are specifi- cally expressed in various cell types. Key regulatory domains of the protein involved in stabilizing the STIM resting state and domains involved in protein activation are described. The focus is on competitive interactions of cytosolic domains of STIM. The recently pub- lished functional data as well as structural data from nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations have been essential in advancing and complementing the characterization of STIM [15,21,22,30]. Moreover, we report results of ongoing functional investigations that are based on these novel NMR data. 2. STIM Proteins STIM is a dimeric type I single-pass TM protein which is mainly anchored in the ER membrane [5,31–33] and to some extent in acidic stores [34] as well as the PM [35–37]. It generally consists of an N-terminal ER luminal portion and a larger C-terminal portion in the cytosol that are connected by a TM domain (Figure1)[ 8]. STIM possesses two main functions: on the one hand, it is a precise sensor of the calcium concentration within the ER lumen; on the other hand, it couples to and gates calcium-selective Orai channels in the plasma membrane [6,7]. In order to perform these two tasks, STIM is equipped with several specialized domains spread across its N- and C-terminal portions [7,38]. There are two homologous STIM proteins called STIM1 and STIM2, each having different isoforms that have been characterized since the discovery of the protein family. For STIM1, these include STIM1 Long (STIM1L) and the recently discovered STIM1A (Figure1a) [ 39,40]. Two studies by Miederer et al. and Rana et al. in 2015 revealed a total of three STIM2 isoforms: STIM2.1 (or STIM2β), STIM2.2 (or STIM2α), and STIM2.3 (Figure1b) [ 41,42]. In this nomenclature, the conventional isoform of STIM2 is termed STIM2.2 and will be used hereafter. All isoforms will be discussed in detail after a comprehensive introduction to STIM proteins that includes the latest developments in the field and a brief report of ongoing functional investigations. Int. J. Mol. Sci. 2021, 22, 378 3 of 18 Figure 1. Domain structure of stromal interaction molecule (STIM) proteins. (a) Primary structure of STIM1 and its isoforms STIM1 Long (STIM1L) and STIM1A. Functionally relevant domains within the endoplasmic reticulum (ER) luminal portion include the canonical (cEF) and noncanonical (nEF) EF hands as well as the sterile alpha motif (SAM). Downstream of the transmembrane domain (TM), the cytosolic portion contains three coiled coil (CC) domains commonly known as CC1, CC2, and CC3 with CC1 being further subdivided into α1, α2, and α3. The C-terminal fragment spanning all three CC domains is termed Orai-activating small fragment (OASF). Another fragment comprising CC2 and CC3 is named CRAC-activating domain (CAD) or STIM-Orai-activating region (SOAR). Further C-terminal domains include the inactivation domain (ID or ID-STIM), the microtubule end-binding domain (EB), and the polybasic domain (PBD) at the outermost C-terminus. STIM1L and STIM1A feature the same general structure but each harbor an additional C-terminal domain inserted downstream of the ID domain by alternative splicing. STIM1L thereby includes an actin-binding domain (ABD) while STIM1A possesses an insert designated as domain A. (b) Primary structure of STIM2.2/STIM2α and its isoforms STIM2.1/STIM2β and STIM2.3. For reasons of simplicity, the 87 amino acid N-terminal signal peptide insertion of STIM2.2 and its isoforms was omitted from this display [43–45]. Due to the high degree of similarity between STIM1 and STIM2.2, their functional domains are essentially equivalent. Alternative splicing leads to inclusion of a small 8 amino acid insert (VAASYLIQ) within the CC2 domain of STIM2.1 and to an upstream end of translation in case of STIM2.3, shortening the protein by 148 amino acids.